Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Table 1 Characteristics of the selected 3F proteins and their derivative peptides.

No.	Name	Sequence¹	aa²	Disulfide	K_d(nM)³	Function⁴	IC50⁵(nM)	IC50/EC50⁶(μM)
1	R10-17	SRPRLN GLAPRAIRAQ TPRARTG	70	-	152 ± 10	NC	NC	-
2	R10-15	QLLACR ATRHTLGHNL ACETPAS	70	-	115 ± 9	C and NI	102 ± 13	NI
3	R10-13	QLLACR ATRHTLGHNL TPRARTG	70	-	48 ± 8	C and I	113 ± 7	1.4 ± 0.3
4	R10-14	APLPYT ATRHTLGHNL ACETPAS	70	-	55 ± 6	C and A	164 ± 9	0.02 ± 0.006
5	13-L1	LVCYQLLA S R PGTLETG PDDFTCV	24	C3 and C23	ND	I	ND	10 ± 1.5
6	13-L2	ETCPDDFTGVATRHTLGHNL TQYCS	25	C3 and C24	ND	NI	ND	NI
7	13-L3	ACAIPTPRARTG C	13	C2 and C13	ND	NI	ND	NI
8	14-L1	LVCYAPLPYT PGTLETG PDDFTCV	24	C3 and C23	ND	A	ND	0.03 ± 0.005
9	14-L3	IPACETPAS C	10	C4 and C10	ND	NI	ND	NI

¹Loop sequences of the proteins are for R10-17, 15, 13 and 14. Sequences of individual peptides are shown in full. 13-L1 represents loop 1 of the protein R10-13, bold letters indicate point mutations, and loop sequences are underlined.
²The number of residues in the proteins (e.g., 70) includes 61 residues of the protein, spacer GGS and 6xHis.
³Affinity was measured by Elisa (25). IL-6 was also assayed and the K_d value was found to be 20 ± 5 nM. ND = not determined.
⁴NC = Non-Competitive, C = Competitive, NI = Non-Inhibitory, I = Inhibitory, A = Agonist.
⁵IC50 was determined by ELISA. IL-6 was also assayed and the value was found to be 80 ± 3 nM.
⁶Measured by IL-6 dependent DS-1 proliferation assay. IC50 = concentration for 50% growth inhibition, which for monoclonal antibody was determined to be 1.2 ± 0.15 nM and ED50 = 50% effective concentration, which for IL-6 was found to be 15 ± 2 pM.

ISSN: 1756-6606